This project is a novel investigation of opioid receptors in amphibians. The overall objective is to examine the antinociceptive action of opioids in amphibian as an evolutionary model that will provide insights into the action of opioids in the clinic and on the street. The premise is that understanding the mechanisms of opioid receptor selectivity and activation in earlier-evolved vertebrates will help in the design and understanding of opioid action in humans. Morphine and other opioid drugs produce analgesia in humans and antinociception in animals by direct interaction with opioid receptor proteins located on neurons in the brain and spinal cord. Knowledge of the detailed relationship between the molecular structure of the opioid drug and the molecular conformation of the opioid receptor is essential to understand pharmacological actions of opioids. Whereas mammalian CNS expresses three distinct opioid receptor genes (MOR1, DOR1, KOR1), each capable of mediating antinociception mammals, amphibians appear to mediate the antinociception effects ofmu, 8, and K-selective opioid agonists via a single opioid receptor, termed the 'unireceptor'. Brain and spinal cord tissue homogenates from the Northern grass frog, Rana pipiens, will be used for all studies. The specific aims are: 1) Determine the affinity, receptor density, and selectivity of sites labeled with ji-, K-, S-selective opioid radioligands; 2) Determine affinity, receptor density, and selectivity of sites labeled with highly-selective mu-, K-, delta-opioid antagonist radioligands; and 3) Characterize the intrinsic activity and selectivity of mu-, K-, S-selective opioid agonists by the stimulation of GTPTS35 binding. Preliminary data are presented for each of these specific aims. Additionally as our lab is the sole purveyor of opioid pharmacology using an earlier-evolved vertebrate model, the proposed studies are a unique examination of the functional evolution of opioid receptors. These studies challenge existing paradigms and further develop a new methodology for the study of opioids. Data obtained will be significant contributions to current understanding of the functional evolution of opioid receptors with regard to opioid ligand selectivity. This knowledge will likely contribute to the to understanding of the mechanisms of opioid receptor activation and may help guide the rational development of improved therapies where opioids drugs are used or abused.